Optical scanning systems essentially direct light produced by a resident light source to a sample and measure the light emitted or reflected by the sample. We discuss scanners below in the context of scanning laser microscopes that are used in fluorescent imaging. The inventive mechanism is not, however, limited to use as part of a scanning laser microscope or as part of a fluorescent imaging system.
A fluorescent imaging system examines fluorescence from chemically-tagged biological samples such as cells, proteins, genes and DNA sequences. The samples may be microarrays, which can include thousands of experiments on a single glass microscope slide. Each experiment consists of a spot of, for example, "target" DNA that is chemically bound to a surface of the glass slide. "Probe" DNA, or alternatively RNA, that has been labeled with a fluorophor is introduced to the surface of the slide and is allowed to hybridize with the target DNA. The sample is then optically scanned, using light of a desired wavelength, such that the fluorophor in the respective experiments emits light.
For a given sample, each experiment emits an amount of light that corresponds to the associated fluor density. The fluor density of an experiment depends on the similarity of the particular target DNA and the probe DNA, since complementary molecules have a greater probability of binding than unrelated molecules. A detector measures the intensity of the light emitted by each experiment included in the sample, and the system can then determine the relative degrees of similarity between the target DNA and the probe DNA.
The measurements associated with the sample depend not only on the fluor densities of the experiments but also on the sensitivity of the system. The system sensitivity depends, in turn, on the power of the incident excitation beam and the gain of the detector. The fluorescent imaging systems have relatively large adjustable ranges of sensitivity, to accommodate the extensive ranges of fluor densities. For example, it is common for the systems to have a dynamic range of four orders of magnitude that is adjustable over another four orders of magnitude. The sensitivity is set by adjusting the power of the incident excitation beam or the gain of the detector, or both.
It is difficult to maintain repeatability with systems having such large ranges of adjustability. Accordingly, for applications in which data are compared from one sample measurement to the next or from one system to the next, the accuracy of the sensitivity setting of the system and the overall calibration of the system are important. Changes in the incident power or the detector gain from what is expected at a given sensitivity setting adversely affect normalization of the data, unless the changes can be quantified.
The system controls the power of the incident excitation beam, which is typically produced by a laser, by including a variable attenuator in the beam path. The attenuator is characterized by an index-to-attenuation transfer curve. It is not uncommon, however, for there to be a relatively large discrepancy between the percentage of attenuation at the respective index settings and the transfer curve. Accordingly, there is often a discrepancy in the expected power of the incident excitation beam relative to the setting of the attenuator. Further, the operating characteristics of the laser may vary over time, such that there is a reduction of the nominal power of the excitation beam before attenuation. It is thus desirable to accurately and periodically characterize, or calibrate, the power level associated with each of the settings of the variable attenuator.
The gain of the detector depends in large part on the gain of the included photometric device. Generally, the device is a photo-multiplier tube, or PMT, which has a high sensitivity and adjustable gain. The detector gain is set by adjusting a DC voltage that is the reference for the PMT power supply. The transfer function of the reference voltage-to-PMT gain is grossly non-linear, however, and the operations of the individual PMTs typically vary from the transfer curve. Further, the operations of the PMTs vary with different wavelengths, and thus, vary between channels on a multiple-channel system. Accordingly, the PMT index settings are only rough estimates of the PMT gain. It is thus desirable to accurately characterize or calibrate the operations of the detector at the various PMT index settings.
Certain known prior systems calibrate system sensitivity by including in a sample or in the field of view of the system one or more reference spots with known fluorescence. These systems measure the fluorescent light emitted by the reference spots as well as the light emitted by the experiments, in the same measurement operation, and use the data associated with the reference spots to quantify the system sensitivity.
The reference spots may be made with fluorescent dyes or solid-state fluorescence. The spots made with fluorescent dyes are vulnerable to "photo-bleaching," and thus, the systems that use these spots may make inaccurate calibration measurements based on damaged spots, or may be unable to make calibration measurements for a given sample. The emission characteristics of the reference spots made of solid state fluorescent material vary with changes in the ambient environment, such as changes in temperature. Accordingly, the systems that use them must compensate for the environmental changes, which adds complexity to the system.